Scientific opinion on flavouring group evaluation 74, revision 4 (FGE.74Rev4): Consideration of aliphatic sulphides and thiols evaluated by JECFA (53rd and 61st meeting) structurally related to aliphatic and alicyclic mono‐, di‐, tri‐ and polysulphides wi

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revision 4 (FGE.74Rev4): Consideration of aliphatic

sulphides and thiols evaluated by JECFA (53rd and 61st

meeting) structurally related to aliphatic and alicyclic

mono-, di-, tri- and polysulphides with or without

additional oxygenated functional groups from chemical

group 20 evaluated by EFSA in FGE.08Rev5

Vittorio Silano, Claudia Bolognesi, Laurence Castle, Kevin Chipman, Jean

Pierre Cravedi, Karl-heinz Engel, Paul Fowler, Roland Franz, Konrad Grob,

Rainer Gürtler, et al.

To cite this version:

Vittorio Silano, Claudia Bolognesi, Laurence Castle, Kevin Chipman, Jean Pierre Cravedi, et al..

Sci-entific opinion on flavouring group evaluation 74, revision 4 (FGE.74Rev4): Consideration of aliphatic

sulphides and thiols evaluated by JECFA (53rd and 61st meeting) structurally related to aliphatic and

alicyclic mono-, di-, tri- and polysulphides with or without additional oxygenated functional groups

from chemical group 20 evaluated by EFSA in FGE.08Rev5. EFSA Journal, European Food Safety

Authority, 2018, 16 (3), 1 p. �10.2903/j.efsa.2018.5167�. �hal-02618061�

ADOPTED: 25 January 2018 doi: 10.2903/j.efsa.2018.5167

Scienti

fic Opinion on Flavouring Group Evaluation 74,

Revision 4 (FGE.74Rev4): Consideration of aliphatic

sulphides and thiols evaluated by JECFA (53rd and 61st

meeting) structurally related to aliphatic and alicyclic

mono-, di-, tri- and polysulphides with or without additional

oxygenated functional groups from chemical group 20

evaluated by EFSA in FGE.08Rev5

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF),

Vittorio Silano, Claudia Bolognesi, Laurence Castle, Kevin Chipman, Jean-Pierre Cravedi,

Karl-Heinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer G€urtler, Trine Husøy,

Sirpa K€arenlampi, Maria Rosaria Milana, Karla Pfaff, Gilles Riviere, Jannavi Srinivasan,

Maria de F

atima Tavares Pocßas, Christina Tlustos, Detlef W€olfle, Holger Zorn,

Romualdo Benigni, Leon Brimer, Gerard Mulder, Agneta Oskarsson, Camilla Svendsen,

Jan van Benthem, Maria Anastassiadou, Siiri Saarma and Wim Mennes

Abstract

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to consider evaluations offlavouring substances assessed since 2000 by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), and to decide whether further evaluation is necessary, as laid down in Commission Regulation (EC) No 1565/2000. The present revision of this FGE is on the assessment of recently submitted toxicity data on methyl propyl trisulfide [FL-no: 12.020], being the representative for a group of seven additional_{flavouring substances: diallyl} trisul_{fide [FL-no: 12.009], dimethyl trisulfide [FL-no: 12.013], dipropyl trisulfide [FL-no: 12.023], methyl} allyl trisul_{fide [FL-no: 12.045], diallyl polysulfides [FL-no: 12.074], methyl ethyl trisulfide [FL-no: 12.155]} and diisopropyl trisulphide [FL-no: 12.280]. Specifications have been provided for all substances. The Panel decided that the 90-day study submitted for [FL-no: 12.020] can be considered only once it is clearly demonstrated that the material tested is representative of the material of commerce and that potential reaction products of the components are not of safety concern. Therefore, no conclusion on the safety of the eight flavouring substances [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.155 and 12.280] can be reached. For methyl-4-oxopentane-thiol [FL-no: 12.169] and 2-mercapto-2-methylpentan-1-ol [FL-no: 12.241], additional subchronic toxicity data are required. The remaining nine substances [FL-no: 12.088, 12.179, 12.198, 12.212, 12.238, 12.239, 12.255, 12.257 and 12.291] in this FGE are not considered of safety concern under the intended conditions of use. © 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

Keywords: _{flavourings, FGE.08, FGE.74, JECFA, sulphides, thiols}

Requestor: European Commission

Question numbers: EFSA-Q-2016-00720, 00721, 00722, 00723, 00724, 00725, 00726, 00727 Correspondence: fip@efsa.europa.eu

Panel members: Claudia Bolognesi, Laurence Castle, Kevin Chipman, Jean-Pierre Cravedi, Karl-Heinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer G€urtler, Trine Husøy, Sirpa K€arenlampi, Wim Mennes, Maria Rosaria Milana, Karla Pfaff, Gilles Riviere, Jannavi Srinivasan, Maria de Fatima Tavares Pocßas, Vittorio Silano, Christina Tlustos, Detlef W€olfle and Holger Zorn.

Acknowledgements: The Panel wishes to thank the hearing experts Vibe Beltoft and Karin Nørby and EFSA staff Annamaria Rossi for the support provided to this scienti_{fic opinion.}

Suggested citation: EFSA CEF Panel (EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids), Silano V, Bolognesi C, Castle L, Chipman K, Cravedi J-P, Engel K-H, Fowler P, Franz R, Grob K, G€urtler R, Husøy T, K€arenlampi S, Milana MR, Pfaff K, Riviere G, Srinivasan J, Tavares Pocßas MF, Tlustos C, W€olfle D, Zorn H, Benigni R, Brimer L, Mulder G, Oskarsson A, Svendsen C, van Benthem J, Anastassiadou M, Saarma S and Mennes W, 2018. Scientific Opinion on Flavouring Group Evaluation 74, Revision 4 (FGE.74Rev4): Consideration of aliphatic sulphides and thiols evaluated by JECFA (53rd and 61st meeting) structurally related to aliphatic and alicyclic mono-, di-, tri- and polysulphides with or without additional oxygenated functional groups from chemical group 20 evaluated by EFSA in FGE.08Rev5. EFSA Journal 2018;16(3):5167, 58 pp.https://doi.org/10.2903/j.efsa.2018.5167 ISSN: 1831-4732

© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made.

The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union.

Table of contents

Abstract... 1

1. Introduction... 4

1.1. Background and Terms of Reference as provided by the requestor... 4

1.1.1.Background... 4

1.1.2.Terms of Reference... 4

1.2. Interpretation of the Terms of Reference... 4

2. Assessment... 4

2.1. History of the evaluation of the substances in the present FGE... 6

2.2. Presentation of the substances in the JECFAflavouring group... 6

2.2.1.Description... 6 2.2.2.Isomers... 9 2.2.3.Specifications... 9 3. Intake estimation... 9 3.1. Status... 9 3.2. EFSA considerations... 9 4. Genotoxicity... 14

4.1. Genotoxicity studies– text taken from the JECFA Report (JECFA, 2000,2004b) ... 14

4.2. Genotoxicity studies– text taken from EFSA FGE.08Rev5 (EFSA CEF Panel, 2012b)... 14

4.3. Genotoxicity study on 2-methyl-4-oxopentane-2-thiol [FL-no: 12.169] from FGE.91Rev2 (EFSA CEF Panel, 2014) ... 15

4.4. EFSA Considerations... 16

5. Evaluation of the additional data submitted for the tri- and polysulphides in FGE.74... 16

6. Short-term and subchronic toxicity... 17

6.1. A 14-day range-finding study with methyl propyl trisulfide [FL-no: 12.020] mixed in the diet (Bauter, 2015a)... 17

6.2. A 14-day rangefinding study with methyl propyl trisulfide [FL-no: 12.020] by oral gavage (Bauter, 2015b).... 17

6.3. A 90-day study by oral gavage with [FL-no: 12.020]... 17

6.4. EFSA considerations... 18

7. Application of the Procedure... 18

7.1. Application of the Procedure to 19 aliphatic sulphides and thiols evaluated by the JECFA (JECFA, 2000, 2004b) ... 18

7.2. Application of the Procedure to aliphatic and alicyclic mono-, di-, tri-, and polysulphides with or without additional oxygenated functional groups evaluated by EFSA in FGE.08Rev5 (EFSA CEF Panel, 2012b) ... 19 7.3. EFSA Considerations relevant for FGE.74... 21

8. Conclusions... 23

Documentation provided to EFSA... 24

References... 26

Abbreviations... 28

Appendix A– Procedure of the safety evaluation... 30

Appendix B– Exposure estimate... 32

Appendix C– Summary of the genotoxicity data... 35

Appendix D– Summary of toxicity data... 41

1.

Introduction

1.1.

Background and Terms of Reference as provided by the requestor

1.1.1.

Background

The use of flavourings is regulated under Regulation (EC) No 1334/2008 of the European Parliament and Council of 16 December 20081 on flavourings and certain food ingredients with flavouring properties for use in and on foods. On the basis of Article 9(a) of this Regulation, an evaluation and approval are required for flavouring substances.

The Union list of flavourings and source materials was established by Commission Implementing Regulation (EC) No 872/20122_{. The list contains flavouring substances for which the scientific} evaluation should be completed in accordance with Commission Regulation (EC) No 1565/20003_.

In May 2014 the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids adopted the opinion on the Flavouring Group Evaluation 74 Revision.3, (FGE.74rev.3) and concluded that further data are required for the tri- and polysulphides substances with Flavis numbers: [FL-nos: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.155 and 12.280] belonging to subgroup VI of this Group.

It also indicated that for these eight substances in subgroup VI (acyclic tri- and polysulphides) [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.155 and 12.280], 90-day studies were available on [FL-no: 12.009 and 12.023], but the studies were not considered adequate for deriving a NOAEL (Morgareidge and Oser, 1970a,b). There were no data on stability of test substances and no results reported from histopathological examinations. The Panel also concluded that tri- and poly-sulphides cannot be covered by NOAELs for dipoly-sulphides, due to the formation of more reactive metabolites than is the case for the disulphides.

On 19 July 2016 the Industry submitted a new dossier with safety studies concerning the substances in this subgroup addressing this issue.

1.1.2.

Terms of Reference

The European Commission requests the European Food Safety Authority (EFSA) to evaluate this new information and, depending on the outcome, proceed to the full evaluation on these _flavouring substances in accordance with Commission Regulation (EC) No 1565/2000.

1.2.

Interpretation of the Terms of Reference

The European Commission requested EFSA to carry out a safety assessment on the substances diallyl trisulfide [FL-no: 12.009], dimethyl trisulfide [FL-no: 12.013], methyl propyl trisulfide [FL-no: 12.020], dipropyl trisulfide [FL-no: 12.023], methyl allyl trisulfide [FL-no: 12.045], diallyl polysulfides [FL-no: 12.074] and methyl ethyl trisulfide [FL-no: 12.155] and diisopropyl trisulphide [FL-no: 12.280] in accordance with Commission Regulation (EC) No 1565/2000.

2.

Assessment

The approach used by EFSA for safety evaluation of flavouring substances is referred to in Commission Regulation (EC) No 1565/2000, hereafter named the ‘EFSA Procedure’. This Procedure is based on the opinion of the Scientific Committee on Food (SCF, 1999), which has been derived from the evaluation procedure developed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 1995, 1996, 1997, 1999a), hereafter named the ‘JECFA Procedure’. The CEF Panel compares the JECFA evaluation of structurally related substances with the result of a corresponding EFSA evaluation, focussing on specifications, intake estimations and toxicity data, especially genotoxicity

1 _{Regulation (EC) No 1334/2008 of the European Parliament and of the Council of 16 December 2008 onflavourings and certain}

food ingredients with flavouring properties for use in and on foods and amending Council Regulation (EEC) No 1601/91, Regulations (EC) No 2232/96 and (EC) No 110/2008 and Directive 2000/13/EC. OJ L 354, 31.12.2008, p. 34–50.

2 _{Commission implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list offlavouring substances provided}

for by Regulation (EC) No 2232/96 of the European Parliament and of the Council, introducing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) No 1565/2000 and Commission Decision 1999/217/EC. OJ L 267, 2.10.2012, p. 1–161.

3 _{Commission Regulation No 1565/2000 of 18 July 2000 laying down the measures necessary for the adoption of an evaluation}

data. The evaluations by EFSA will conclude whether the flavouring substances are of no safety concern at their estimated levels of intake, whether additional data are required or whether certain substances should not be put through the EFSA Procedure.

The following issues are of special importance. Intake

In its evaluation, the Panel as a default uses the ‘maximised survey-derived daily intake’ (MSDI) approach to estimate the per capita intakes of theflavouring substances in Europe.

In its evaluation, JECFA includes intake estimates based on the MSDI approach derived from both European and USA production figures. The highest of the two MSDI figures is used in the evaluation by JECFA. It is noted that in several cases, only the MSDI _{figures from the USA were available,} meaning that certain _{flavouring substances have been evaluated by JECFA only on the basis of these} figures. For substances in the Union List of flavouring substances2 _{for which this is the case, the} Panel will need the European Union (EU) production figures in order to finalise the evaluation.

When the Panel examined the information provided by the European Flavour Industry on the use levels in various foods, it appeared obvious that the MSDI approach in a number of cases would grossly underestimate the intake by regular consumers of products flavoured at the use level reported by industry, especially in those cases where the annual production values were reported to be small. In consequence, the Panel had reservations about the data on use and use levels provided and the intake estimates obtained by the MSDI approach. It is noted that JECFA, at its 65th meeting considered ‘how to improve the identification and assessment of flavouring agents, for which the MSDI estimates may be substantially lower than the dietary exposures that would be estimated from the anticipated average use levels in foods’ (JECFA, 2006).

In the absence of more accurate information that would enable the Panel to make a more realistic estimate of the intakes of the _{flavouring substances, the Panel has decided also to perform an} estimate of the daily intakes per person using a modi_{fied ‘theoretical added maximum daily intake’} (mTAMDI) approach based on the normal use levels reported by industry.

As information on use levels for theflavouring substances has not been requested by JECFA or has not otherwise been provided to the Panel, it is not possible to estimate the daily intakes using the mTAMDI approach for many of the substances evaluated by JECFA. The Panel will need information on use levels in order tofinalise the evaluation.

Threshold of 1.5 _{lg/person per day (Step B5) used by JECFA}

JECFA uses the threshold of concern of 1.5lg/person per day as part of the evaluation procedure: The Committee noted that this value was based on a risk analysis of known carcinogens which involved several conservative assumptions. The use of this value was supported by additional information on developmental toxicity, neurotoxicity and immunotoxicity. In the judgement of the Committee, flavouring substances for which insufficient data are available for them to be evaluated using earlier steps in the Procedure, but for which the intake would not exceed 1.5 lg/person per day would not be expected to present a safety concern. The Committee recommended that the Procedure for the Safety Evaluation of Flavouring Agents used at the 46th meeting be amended to include the last step on the right-hand side of the original Procedure (_{‘Do the condition of use result in an intake} greater than 1.5 lg per day?’) (JECFA, 1999a).

In line with the opinion expressed by the Scientific Committee on Food (SCF, 1999), the Panel does not make use of this threshold of 1.5 lg/person per day.

Genotoxicity

As reflected in the opinion of SCF (1999), the Panel has in its evaluation focussed on a possible genotoxic potential of the flavouring substances or of structurally related substances. Generally, substances for which the Panel has concluded that there is an indication of genotoxic potential in vitro, will not be evaluated using the EFSA Procedure until further genotoxicity data are provided. Substances for which a genotoxic potential in vivo has been concluded, will not be evaluated through the Procedure.

Specifications

Regarding speci_{fications, the evaluation by the Panel could lead to a different opinion than that of} JECFA, since the Panel requests information on, e.g. isomerism.

Structural Relationship

In the consideration of the JECFA-evaluated substances, the Panel will examine the structural relationship and metabolism features of the substances within the _{flavouring group and compare this} with the corresponding Flavouring Group Evaluation (FGE).

2.1.

History of the evaluation of the substances in the present FGE

At its 61st meeting, JECFA evaluated a group of 12 flavouring substances consisting of aliphatic sulphides and thiols (JECFA, 2004a). One substance was not in the Register. The remaining 11 flavouring substances have originally been considered by EFSA in FGE.74 (EFSA, 2008). The Panel concluded that for two substances [FL-no: 12.169 and 12.241], the Procedure should not be applied until adequate genotoxicity data become available and for three substances [FL-no: 12.179, 12.198 and 12.212] additional toxicity data were required.

In thefirst revision of Flavouring Group Evaluation 74 (FGE.74Rev1), there was a reassessment of four candidate substances due to subgrouping of the substances based on the type of sulphur-containing functional groups. This is in accordance with what has been done in FGE.08Rev1 (EFSA CEF Panel, 2010a) and in FGE.91 (EFSA CEF Panel, 2010b), which also consider substances with sulphur-containing functional groups. The candidate substances in FGE.74Rev1 that have been reassessed due to this are [FL-no: 12.179, 12.198, 12.212 and 12.280]. The outcome of the evaluation is explained in Section 7. Furthermore, the FGE.74Rev1 included the assessment of seven additional substances [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074 and 12.155] evaluated by JECFA at the 53rd meeting (JECFA, 2000). The reason for the inclusion of these seven substances is explained in Section 2.2.1.

In the second revision of FGE.74, FGE.74Rev2, one candidate substance was added, diallyl sulfide [FL-no: 12.088]. This substance has been evaluated by JECFA at the 53rd meeting (JECFA, 2000). The reason for the inclusion of this substance is explained in Section 2.2.1. For four substances [FL-no: 12.009, 12.020, 12.045 and 12.169] additional information on specifications received after publication of FGE.74Rev1 has been included.

The third revision of FGE.74, FGE.74Rev3, concerned the evaluation of a group of ten substances for which the concern for genotoxicity was alleviated by the Panel. The Panel concluded that the evaluation for [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.155, 12.169, 12.241 and 12.280] could not be finalised at Step B4 of the Procedure due to the lack of a no observed adverse effect level (NOAEL) and that additional toxicity data would, therefore, be required.

FGE Adopted by

EFSA Link

No. of substances FGE.74 January 2008 http://www.efsa.europa.eu/en/efsajournal/pub/987.htm 11 FGE.74Rev1 September 2010 http://www.efsa.europa.eu/en/efsajournal/pub/1842.htm 18 FGE.74Rev2 November 2011 http://www.efsa.europa.eu/en/efsajournal/pub/2458.htm 19 FGE.74Rev3 May 2014 https://www.efsa.europa.eu/it/efsajournal/pub/3710.htm 19 FGE.74Rev4 January 2018 http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2018.5167/full 19

FGE: Flavouring Group Evaluation; EFSA: European Food Safety Authority.

The present revision of FGE.74, FGE.74Rev4, concerns the consideration of seven candidate substances [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074 and 12.155] from the 53rd meeting of JECFA and one candidate substance [FL-no: 12.280] from the 61st meeting of JECFA based on new toxicity data submitted on the representative substance methyl propyl trisulfide [FL-no: 12.020]. Additionally, normal and maximum use levels and updated production volumes have become available by the Flavour Industry for 18 substances: [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.088, 12.155, 12.169, 12.179, 12.198, 12.212, 12.238, 12.239, 12.241, 12.555, 12.257 and 12.280] (EFFA, 2017). The updated production volumes have been used for calculation of MSDI values.

2.2.

Presentation of the substances in the JECFA

flavouring group

2.2.1.

Description

JECFA status

JECFA has evaluated a group of 12flavouring substances consisting of aliphatic sulphides and thiols at the 61st meeting (JECFA, 2004a,b).

JECFA has at the 53rd meeting (JECFA, 2000), before 2000, evaluated a group of 137 flavouring substances consisting of aliphatic and aromatic sulphides and thiols with and without an additional oxygenated functional group.

EFSA considerations

This FGE deals with 19 JECFA-evaluated substances. Eleven substances from the 61st meeting, 2003, and eight substances from the 53rd meeting, 1999, because:

•

Of the 12 aliphatic sulphides and thiols evaluated by JECFA at the 61st meeting, one is not in the Register (spiro[2,4-dithia-1-methyl-8-oxabicyclo(3.3.0)octane-3,30-(10-oxa-20-methyl)-cyclopentane], JECFA-no: 1296). From the 61st JECFA meeting, 11 substances remain to be evaluated by EFSA.

•

Of the 137 aliphatic and aromatic sulphides and thiols with and without an additional oxygenated functional group evaluated by JECFA at the 53rd meeting, seven are acyclic polysulphides [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074 and 12.155]. These seven substances were evaluated by JECFA before the year 2000 and have been used as supporting substances in FGE.08 and following revisions. Forflavouring substances evaluated by JECFA before 2000, it is laid down in Commission Regulation (EC) No 1565/20003 that if they are considered acceptable at the current estimated intake by JECFA and comply with the general use criteria, they could be included in the list of authorised substances without undergoing a separate evaluation for the time being. In the FGE.08Rev1 (EFSA CEF Panel, 2010a), it was recognised that tri- and polysulphides may form reactive metabolites and accordingly in FGE.74Rev1, the Panel decided to reconsider these seven polysulphides previously evaluated by JECFA (see Comment on subgroup VI (acyclic tri- and polysulphides) below). Further, for diallyl sulfide [FL-no: 12.088], which JECFA evaluated at Step B5, no NOAEL exists to provide a margin of safety (MoS). However, as the estimated intake in the USA of 0.4 lg/capita per day is below the threshold of concern of 1.5 lg/person per day, the JECFA Committee noted that intakes below this value would not be expected to present a safety concern. In line with the opinion expressed by the SCF (1999), the Panel does not make use of this threshold of 1.5lg/person per day. From the 53rd JECFA meeting, eight substances remain to be evaluated by EFSA. In addition, in FGE.08Rev1, the genotoxicity issues that were noted for candidate tertiary thiols are obviously also of relevance for two candidate JECFA-evaluated tertiary thiols [FL no: 12.169 and 12.241] in this consideration.

The Panel concluded that the substances in the JECFA flavouring group of aliphatic sulphides and thiols are structurally related to the group of aliphatic and alicyclic mono-, di-, tri- and polysulphides with or without additional oxygenated functional groups evaluated by EFSA in the Flavouring Group Evaluation 08, Revision 5 (FGE.08Rev5). Depending on the type of sulfur-containing functional groups, the substances in FGE.08Rev5 were subdivided into 11 subgroups:

I Acyclic sulphides II Cyclic sulphides

III Monothiols, including tertiary monothiols IV Dithiols

V Acyclic and cyclic disulphides VI Acyclic polysulphides

VII Mono-, di-, tri- and polysulphides with thioacetal structure VIII Thioesters

IX Thioic acid

X Sulphoxides/sulphones and sulphonates XI Cyclic thioketal fused with an oxolane ring.

In the following part of this fourth revision of FGE.74 (FGE.74Rev4), there will be reference to the fifth revision of FGE.08 (FGE.08Rev5) (EFSA CEF Panel, 2012b). It is also the fifth revision of FGE.08 that is used in the application of the Procedure by EFSA (Section 7.2of this FGE.74Rev4).

The 19 JECFA-evaluated substances in the present FGE will be considered in compliance with these EFSA defined subgroups.

Comment on Subgroup VI (acyclic tri- and polysulphides)

During the evaluation of the candidate substances in the FGE.08Rev1 (EFSA CEF Panel, 2010a), it was recognised that tri- and polysulphides (subgroup VI) may form reactive metabolites through reaction with endogenous thiols forming a thiol and a hydropersulphide or perthiol. Compared to thiols, perthiols may be strong reducing agents, forming reactive products when exposed to oxidants. Based on the above information it was concluded that tri- and polysulphides could not be covered by NOAELs for disulphides, due to the formation of more reactive metabolites.

The Panel noted that in FGE.08Rev1 seven supporting substances are tri- or polysulphides [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074 and 12.155]. These substances were evaluated by JECFA before the year 20004 (accepted at Step B4 based on NOAELs derived from studies with disulphides), and therefore at first not included in the consideration performed by EFSA on the JECFA-evaluated substances in FGE.74.

Accordingly, the decision taken in FGE.08Rev1 has had an impact on the tri- and polysulphides in FGE.74 (one substance [FL-no: 12.280]) as well as those evaluated by JECFA at its 53rd meeting, before 2000 (seven substances [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074 and 12.155]), which were therefore included in thefirst revision of FGE.74 (FGE.74Rev1) (EFSA CEF Panel, 2010c). Distribution of the FGE.74Rev4 substances into subgroups

The 19 JECFA-evaluated substances in this FGE have been assigned to _{five subgroups, in} accordance with the subdivision in FGE.08Rev5. This subdivision is shown in Table 1below.

Table 1: Allocation of the 19 JECFA-evaluated substances considered in FGE.74 into subgroups according to subdivision in FGE.08Rev5

FL-no: Register name Structural formula

I Acyclic sulphides

12.088 Diallyl sulfide S

12.179 2-(Methylthio)ethan-1-ol HO S 12.212 Ethyl-5-(methylthio)valerate O S O III Monothiols 12.169 2-Methyl-4-oxopentane-2-thiol SH O 12.238 3-Mercapto-2-methylpentan-1-ol OH SH 12.239 3-Mercapto-2-methylpentanal _O HS 12.241 2-Mercapto-2-methylpentan-1-ol HO SH 12.255 Ethyl 3-mercaptobutyrate O O SH 12.291 3-Mercapto-2-methyl-1-butanol OH SH

V Acyclic and cyclic disulphides

12.198 2,3,5-Trithiahexane S S

S

4

Forflavouring substances evaluated by the JECFA before 2000 it is laid down in Commission Regulation (EC) 1565/2000 that if they are considered acceptable at the current estimated intake by the JECFA and comply with the general use criteria, they could be included in the list of authorised substances without undergoing a separate evaluation for the time being.

2.2.2.

Isomers

JECFA status

Two substances have one chiral centre [FL-no: 12.241 and 12.255] and three substances have two chiral centres [FL-no: 12.238, 12.239 and 12.291] in the group of the JECFA-evaluated sulphides and thiols. EFSA considerations

Adequate information on isomeric composition is available for all the substances in FGE.74Rev4. For the two stereoisomeric substances [FL-no: 12.241 and 12.255] with one chiral centre, the CAS register number (CASrn) is considered to cover the stereoisomeric composition as a racemate.

2.2.3.

Speci

fications

JECFA status

The JECFA specifications are available for all 19 substances (JECFA, 1999b, 2003). See Table 2. EFSA considerations

The available specifications are considered adequate for 19 substances.

3.

Intake estimation

3.1.

Status

For all substances evaluated through the JECFA Procedure production volumes (JECFA, 2000, 2004b), EFSA has received updated production volumes for the EU (EFFA, 2017), based on which MSDI values have been calculated (see Appendix B, TableB.2).

3.2.

EFSA considerations

For all 19 substances, the flavour industry submitted normal and maximum use levels (Flavour Industry, 2008; EFFA, 2017). Based on the normal use levels, the mTAMDI figures have been calculated. For 18 substances [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.155, 12.169, 12.179, 12.198, 12.212, 12.238, 12.239, 12.241, 12.555, 12.257, 12.280 and 12.291], the mTAMDI intake estimates are below the threshold of toxicological concern (TTC) for their structural class. For one substance [FL-no: 12.088] the mTAMDI intake estimate is above the TTC for its structural class.

Use levels and mTAMDI values are presented in AppendixB, TablesB.1 andB.2.

FL-no: Register name Structural formula

VI Acyclic tri- and polysulphides

12.009 Diallyl trisulfide S

S S

12.013 Dimethyl trisulfide S

S S

12.020 Methyl propyl trisulfide

S S S 12.023 Dipropyl trisulfide S S S

12.045 Methyl allyl trisulfide S _S S

12.074 Diallyl polysulfides

SX

X=2,3,4 or 5

12.155 Methyl ethyl trisulfide S _S S

12.280 Diisopropyl trisulphide S S S VIII Thioesters 12.257 Ethyl 4-(acetylthio)butyrate O O S O

Table 2: Specification summary of the substances in FGE.74 Revision 4

FL-no

JECFA-no EU Register name

Structural formula FEMA no CoE no CAS no Phys. form Mol. formula Mol. weight Solubility(a) Solubility in ethanol(b) Boiling point,°C(c) Melting point,°C ID test Assay minimum Refrac. Index(d) Spec. gravity(e) EFSA comments 12.009 587 Diallyl trisulfide S S S 3265 486 2050-87-5 Liquid C6H10S3 178.33 Insoluble Insoluble 112–120 (21 hPa) IR 65% 1.600–1.620 1.135–1.170

Min. assay value 65% (min. 95% allyl di-, tri- and tetrasulfides) (EFFA, 2017); secondary components 20–25% allyl disulfide; 5–7% allylsulfide; 5–7% allyl tetrasulfide (DG SANCO, 2011) 12.013 582

Dimethyl trisulfide S S S ₃₂₇₅ 539 3658-80-8 Liquid C2H6S3 126.26 Very slightly soluble Soluble 165–170 IR 97% 1.595–1.605 1.195–1.210 EFFA (2017) 12.020 584

Methyl propyl trisulfide S S S ₃₃₀₈ 586 17619-36-2 Liquid C4H10S3 154.30 Very slightly soluble Soluble 52 (1.6 hPa) IR 45% 1.558–1.570 1.095–1.101

Also contains 25% dipropyl trisulfide, 12% dipropyl disulfide, 14% dimethyl disulfide, 3% methyl propyl sulfide (EFFA, 2017). More than 95% identified components 12.023 585 Dipropyl trisulfide S S S 3276 726 6028-61-1 Liquid C6H14S3 182.36 Almost insoluble Soluble 98 (5 hPa) IR 99% 1.542–1.590 0.952 Including up to 15% dipropyl disulfide (EFFA, 2017)

12.045 586

Methyl allyl trisulfide S S S 3253 11867 34135-85-8 Liquid C4H8S3 152.29 Very slightly soluble Soluble 47 (1 hPa) NMR 80% 1.593–1.603 0.975–0.985 Min. 10% dimethyl trisulfide; 6–8% allyl trisulfide (EFFA, 2017). More than 95% identified components

FL-no

JECFA-no EU Register name

Structural formula FEMA no CoE no CAS no Phys. form Mol. formula Mol. weight Solubility(a) Solubility in ethanol(b) Boiling point,°C(c) Melting point,°C ID test Assay minimum Refrac. Index(d) Spec. gravity(e) EFSA comments 12.074 588 Diallyl polysulfides SX X=2,3,4 or 5 3533 11912 72869-75-1 Liquid C6H10S2 146.30 Insoluble Slightly soluble 68 (20 hPa) IR NMR 95% 1.643–1.653

1.220 (20°) Mixture of allyl disulfides(2–10%), allyl trisulfides (20–30%), allyl

tetrasulfides (30–40% and allyl pentasulfides

(30–40%). The number of S atoms in the chemical formula varies from 2 to 5 (EFFA, 2017)

12.088 458

Diallyl sulfide S

2042 11846 592-88-1 Liquid C6H10S 114.21 Insoluble Sparingly soluble 138–139 IR 97% 1.488–1.492 0.887–0.892 Solubility in ethanol (EFFA, 2011) 12.155 583

Methyl ethyl trisulfide S

S S ₃₈₆₁ 31499-71-5 Liquid C3H8S3 140.28 Very slightly soluble Soluble 46–47 (5 hPa) NMR 97% 1.510–1.520 0.955–0.965 12.169 1293 2-Methyl-4-oxopentane-2-thiol SH O ₃₉₉₇ 11500 19872-52-7 Liquid C6H12OS 132.23 Soluble Very slightly soluble 47–49 (20 hPa) IR NMR MS 48% 1.431–1.437 1.032–1.037

The Register name to be changed to 4-mercapto-4-methyl-2-pentanone. Min. assay value is 48% and secondary component 4-methyl-3-penten-2-one [FL-no: 07.101] 48-50% (DG SANCO, 2011); supplied as a 1% solution in propylene glycol. More than 95% identified components 12.179 1297 2-(Methylthio)ethan-1-ol HO S 4004 11545 5271-38-5 Liquid C3H8OS 92.16 Insoluble Soluble 169–171 IR NMR MS 98% 1.490–1.498 1.055–1.065 (20°)

FL-no

JECFA-no EU Register name

Structural formula FEMA no CoE no CAS no Phys. form Mol. formula Mol. weight Solubility(a) Solubility in ethanol(b) Boiling point,°C(c) Melting point,°C ID test Assay minimum Refrac. Index(d) Spec. gravity(e) EFSA comments 12.198 1299 2,3,5-Trithiahexane S S S 4021 42474-44-2 Liquid C3H8S3 140.30 Insoluble Soluble 56–58 (10 hPa) MS 95% 1.436–1.444 1.157–1.163 12.212 1298 Ethyl-5-(methylthio) valerate O S O 3978 233665-98-0 Liquid C8H16O2S 176.27 Insoluble Soluble 227 IR NMR MS 96% 1.460–1.464 0.993–1.003 (20°) Register name to be changed to ethyl 5-(methylthio)valerate 12.238 1291 3-Mercapto-2-methylpentan-1-ol OH SH 3996 227456-27-1 Liquid C6H14OS 134.24 Slightly soluble Soluble 50 (0.7 hPa) IR NMR 99% 1.480–1.490 0.985–0.995 Mixture of four diastereoisomers, each about 25% (EFFA, 2014) 12.239 1292 3-Mercapto-2-methylpentanal O HS 3994 227456-28-2 Liquid C6H12OS 132.23 Insoluble Soluble 98–100 (13 hPa) IR 96% 1.523–1.529 1.095–1.103 Mixture of four diastereoisomers, each about 25% (EFFA, 2014) 12.241 1290 2-Mercapto-2-methylpentan-1-ol HO SH 3995 258823-39-1 Liquid C6H14OS 134.24 Slightly soluble Soluble 57–59 (0.8 hPa) IR NMR 99% 1.476–1.483 0.968–0.974 (20°) Racemate. CASrn is considered to cover the stereoisomeric composition as racemate 12.255 1294 Ethyl 3-mercaptobutyrate O O SH 3977 156472-94-5 Liquid C6H12O2S 148.22 Insoluble Soluble 188 IR NMR MS 97% 1.448–1.453 1.011–1.021 (20°) Racemate. CASrn is considered to cover the stereoisomeric composition as racemate 12.257 1295 Ethyl 4-(acetylthio)-butyrate O O S O 3974 104228-51-5 Liquid C8H14O3S 190.26 Insoluble Soluble 262 IR NMR MS 96% 1.468–1.472 1.073–1.083 (20°) 12.280 1300 Diisopropyl trisulphide S S S 5943-34-0 Liquid C6H14S3 182.40 Insoluble Soluble 107–108 (13 hPa) NMR MS 95% 1.441–1.445 1.134–1.140 EFFA (2017)

FL-no

JECFA-no EU Register name

Structural formula FEMA no CoE no CAS no Phys. form Mol. formula Mol. weight Solubility(a) Solubility in ethanol(b) Boiling point,°C(c) Melting point,°C ID test Assay minimum Refrac. Index(d) Spec. gravity(e) EFSA comments 12.291 1289 3-Mercapto-2-methyl-1-butanol OH SH ₃₉₉₃ 227456-33-9 Liquid C5H12OS 120.21 Slightly soluble Freely soluble 98 (at 2.7 hPa) IR NMR MS 98% 1.482–1.490 1.002–1.008 Mixture of four diastereoisomers, each about 25% (EFFA, 2014)

FL-no.: Flavour Information System number; JECFA: The Joint FAO/WHO Expert Committee on Food Additives; FEMA: Flavor and Extract Manufacturers Association; CoE: Council of Europe; CAS: Chemical Abstract Service; ID: Identity; IR: infrared spectroscopy; NMR: nuclear magnetic resonance; MS: mass spectrometry; CASrn: Chemical Abstract Service register number. (a): Solubility in water, if not otherwise stated.

(b): Solubility in 95% ethanol, if not otherwise stated. (c): At 1,013.25 hPa (1 atm), if not otherwise stated. (d): At 20°C, if not otherwise stated.

4.

Genotoxicity

4.1.

Genotoxicity studies

– text taken

5

from the JECFA Report (JECFA,

2000, 2004b)

The reverse mutation test was performed for diallyl sulfide [FL-no: 12.088] (0.004–0.44 lg/mL), using Salmonella Typhimurium strain TA100. No genotoxicity was observed (Eder et al., 1982).6

Groups of male ICR mice were given two doses 48 h apart of a mixture containing diallyl sulfide [FL-no: 12.088], allyl disulfide (JECFA-no: 572) or diallyl trisulfide [FL-no: 12.009] in corn oil at doses of 10 or 20 mg/mL by gavage. The doses were estimated to provide 0.33 or 0.67 mmol/kg bw or 50 or 100 mg/kg bw on the basis of the composition of the mixture. No increase in the frequency of micronucleated polychromatic erythrocytes was seen in bone marrow cells (Marks et al., 1992).

Erythro- and threo-3-mercapto-2-methylbutanol [FL-no: 12.291 (3-mercapto-2-methyl-1-butanol)] (50–5,000 lg/plate) was evaluated for mutagenic activity in the modified Ames test with pre-incubation in the presence and absence of metabolic activation in S. Typhimurium strains TA97, TA98, TA100, TA102 and TA1535. No genotoxic effects were observed (Gocke, 1997).

For a summary of in vitro/in vivo genotoxicity data considered by JECFA, see AppendixC, TableC.1.

4.2.

Genotoxicity studies

– text taken

7

from EFSA FGE.08Rev5 (EFSA

CEF Panel, 2012b)

In vitro/in vivo

Genotoxicity in vitro data are available for three candidate substances: di-(1-propenyl)-sulfide (mixture) [FL-no: 12.298] (subgroup I), 2-methylpropane-2-thiol [FL-no: 12.174] (subgroup III) and dibutyl disul_{fide [FL-no: 12.111] (subgroup V). In addition studies are available on 11 supporting} substances from subgroups I (1), III (4), V (4) and VIII (2).

In vivo data are available for three supporting substances from subgroups I (1), III (1) and V (1). Subgroup I (Acyclic sulphides)

In vitro data are available for the candidate substance, di-(1-propenyl)-sulfide [FL-no: 12.298]; Ames test: S. Typhimurium TA98, TA100, TA102, TA1535, TA1537, 1–100 lg/plate. Result was negative with and without metabolic activation (Stien, 2005).

For supporting substances, only data on diallyl sulfide [FL-no: 12.088] are available. Diallyl sulfide was negative in a limited bacterial reversion assay using one strain only (TA100) and provided equivocal results in an in vitro cytogenetic test in which increased incidences of cells with chromosomal aberrations and sister chromatid exchanges (SCEs), statistically significant but not dose related, were observed. In vivo diallyl sulfide was evaluated as negative in a micronucleus test in mouse bone marrow, which was, however, not designed to evaluate the genotoxicity of the substance itself as it was tested in a mixture. Overall the data available do not allow evaluation of the genotoxicity of the substances of this subgroup.

Subgroup III (Monothiols)

2-Methylpropane-2-thiol [FL-no: 12.174] is reported to be negative in an Ames test. It is reported to be positive in a mouse lymphoma assay without metabolic activation and negative in the test with metabolic activation, and it is reported to be negative in an in vitro SCE assay. However, these studies are reported only as summaries (Phillips Petroleum Company, 1990a). Some details are available for methods but not for the results. Although the validity of these studies cannot be fully evaluated, the positive result in the mouse lymphoma assay raises concern with respect to the potential for genotoxicity of this tertiary thiol and structurally related compounds, i.e. candidate substance 2-methylbutane-2-thiol [FL-no: 12.172] and ethyl-2-mercapto-2-methyl propanoate [FL-no: 12.304] and the five supporting substances [FL-no: 12.038, 12.085, 12.137, 12.138 and 12.145].

5 _{The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE}

has been removed.

6 _{The Panel noted that the publication of Eder et al., 1982 is not the correct paper to quote from. It has not been possible for}

EFSA to identify the correct paper.

7 _{The text is taken from the indicated reference source, but text related to subgroups not included in the present FGE has been}

The in vitro data available for the other substances in this subgroup do not provide indication of concern for genotoxicity.

Subgroup V (Acyclic and Cyclic disulphides)

Dibutyl disulfide [FL-no: 12.111] is reported to be negative in a mouse lymphoma assay (Dooley et al., 1987). However, the study is reported only as an abstract, and thus, the validity cannot be evaluated.

Further data are available for the supporting substances diallyl disulfide [FL-no: 12.008], dimethyldisulfide [FL-no: 12.026], phenyl disulfide [FL-no: 12.043] and benzyl disulfide [FL-no: 12.081]. All substances were reported to be negative in the Ames test. In addition, diallyl disulfide was reported to be positive in a chromosomal aberration assay in vitro, with and without metabolic activation, and weakly positive in a SCE assay. However, the validity of these _{findings is doubtful as} chromosomal aberrations were only increased in conditions associated with extensive (_{> 90%)} lethality, and because of the limitation of SCE in genotoxic hazard identification.

Subgroup VI (Acyclic tri- and polysulphides)

No genotoxicity information of sufficient quality is available. Subgroup VIII (Thioesters)

The in vitro data available on supporting substances provide no indication of concern for genotoxicity.

Conclusion on genotoxicity

Most in vitro and in vivo studies are of limited or insufficient quality and provide only limited information.

The available data raise concern with respect to genotoxicity of three tertiary thiols [FL-no: 12.172, 12.174 and 12.304], included as candidate substances in subgroup III. Hydrolysis of the candidate substance 2,4,4-trimethyl-1,3-oxathiane [FL-no: 16.057], included in subgroup VII, leads to the formation of a tertiary thiol structurally related to the above-mentioned compounds. Therefore, there is also concern with respect to genotoxicity of this candidate substance. The Panel noted that in FGE.08 five of the supporting substances were tertiary thiols [FL-no: 12.038, 12.085, 12.137, 12.138 and 12.145] for which a concern for genotoxicity has been raised in the FGE.08Rev1. These supporting substances have been evaluated by JECFA at the 53rd meeting (JECFA, 2000JECFA, 2000, 2004b). These supporting substances have been considered by EFSA in FGE.91 (EFSA CEF Panel, 2010b).

In addition, genotoxicity of the candidate substance methyl methanethiosulfonate [FL-no: 12.159], included in subgroup X, could not be assessed from the data available. However, due to the similarity with methyl methanesulfonate, a direct acting mutagen and carcinogen, there is concern with respect to genotoxic potential of this candidate substance.

Therefore, the Panel decided that the Procedure could not be applied to the candidate substances [FL-no: 12.159, 12.172, 12.174, 12.304 and 16.057] until adequate in vivo genotoxicity data become available.

The other in vitro/in vivo genotoxicity data available, often from limited or poorly reported studies do not provide clear indication of concern for genotoxicity for the remaining candidate substances included in the present evaluation.

For a summary of in vitro/in vivo genotoxicity data considered by EFSA, see AppendixC, TablesC.2 and C.3of this FGE.

4.3.

Genotoxicity study on 2-methyl-4-oxopentane-2-thiol [FL-no:

12.169] from FGE.91Rev2 (EFSA CEF Panel, 2014)

In vitro

2-Methyl-4-oxopentane-2-thiol [FL-no: 12.169] was tested in S. Typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence or absence of S9-mix (Mc Garry, 2012). In the first experiment, the concentrations tested were 5.0, 15.8, 50.0, 158.1, 500.0, 1,581 and 5,000 lg/plate, and the plate incorporation method was used. No evidence of toxicity was observed in the absence or presence of S9-mix in any tester strains. In the second experiment, the concentrations were 156.3, 312.5, 625.0, 1,250, 2,500 and 5,000 lg/plate of 2-methyl-4-oxopentane-2-thiol, and treatments in the presence of S9-mix used the pre-incubation method. Evidence of toxicity was observed through slight thinning of the background lawn and/or marked reduction in revertant numbers in all strains at

2,500 and/or 5,000 lg/plate in the presence of S9-mix and in TA1537 in the absence of S9-mix. Thus, the study design complied with current recommendations and an acceptable top concentration was achieved. There was no evidence of any mutagenic effect induced by 2-methyl-4-oxopentane-2-thiol in any of the strains, either in the absence or presence of S9-mix.

For a summary of the genotoxicity data on 2-methyl-4-oxopentane-2-thiol, see AppendixC, Table C.4.

4.4.

EFSA Considerations

Subgroup III includes the tertiary thiols for which a genotoxicity concern was established based on data from a limited gene mutation assay for candidate substances in FGE.08Rev1 [FL-no: 12.174] and additional genotoxicity data were requested for this group of substances. Since the publication of the latest revision of FGE.08, FGE.08Rev5, the Industry has submitted a new bacterial mutation assay for the tertiary thiol [FL-no: 12.169] included in FGE.74. This substance is considered by the Panel to be representative for the whole group of tertiary thiols (in FGE.08, FGE.74 and FGE.91). Based on the new genotoxicity data, the Panel concluded that 2-methyl-4-oxopentane-2-thiol [FL-no: 12.169] was not genotoxic in the assay and that 2-methyl-4-oxopentane-2-thiol [FL-no: 12.169] and 2-mercapto-2-methylpentan-1-ol [FL-no: 12.241] do no longer give rise to concern with respect to gene mutations. Therefore, these two substances can be evaluated using the Procedure in the present FGE. The Panel noted that of the material of commerce for [FL no: 12.169], approximately half consists of the a, b-unsaturated carbonyl, 4-methyl-3-penten-2-one [FL-no: 07.101], for which concern for genotoxicity was ruled out in FGE.204 (EFSA CEF Panel, 2012a) and evaluated using the Procedure in FGE.63Rev2 (EFSA CEF Panel, 2013).

Although the available data are limited8 the Panel considered that for the 19 substances in FGE.74Rev4 the genotoxicity data do not preclude evaluating these substances through the Procedure.

5.

Evaluation of the additional data submitted for the tri- and

polysulphides in FGE.74

In the earlier EFSA-evaluations of acyclic tri- and polysulphides (subgroup VI in FGE.08), the evaluation stopped at Step B4 due to lack of NOAEL for a representative substance. The available 90-day feeding studies on dipropyl trisulfide and diallyl trisulfide in rats by Morgareidge and Oser (1970a, b) were not considered sufficient to derive a NOAEL.

The shortcomings of the Morgareidge and Oser studies are the following:

•

No data on the stability of the test substance in feed are given.

•

There are no histopathology data.

•

Nearly all animals, including control animals, were affected by inflammatory changes in respiratory tract, and in other organs (mainly liver). These changes (probably caused by infections) prohibit adequate interpretation of the study results.

•

The data on haematology, clinical chemistry and urine analysis (performed for eight animals in the test-substance groups respectively, and eight animals in the control group at weeks 6 and 12) are only shown as a mean for the three groups, and without any indication of variation between the individuals (e.g. no SD, etc.)

Data submitted on the metabolism and structure–activity relationships of sulphides were mainly in accordance with the information provided in FGE.08 and FGE.74.

To support a potential read-across, the flavour industry submitted a discussion with the aim to show that NOAELs from toxicity studies for sulphides, disulphides and for tri- and polysulphides are of the same magnitude. In this discussion, route to route extrapolation from inhalatory to oral was applied, but the technique to accomplish this has been shown to be inadequate (Rennen et al., 2004). Additionally, the oral long-term studies presented were one-dose-level-only and consequently of limited relevance to judge whether substances are equipotent or not. The discussion on magnitude of NOAELs was, therefore, not considered relevant. The Panel concluded that the extrapolations made by the applicant cannot be used to support derivation of a NOAEL for the oral route.

In FGE.74Rev3, the Panel concluded that the additional information as submitted by the flavour industry (IOFI, 2013) was insuf_{ficient to evaluate the safety of the tri- and polysulphides and that a}

8 _{The Panel noted that an in vivo micronucleus study is available on [FL-no: 12.169], which will be considered in the next}

90-day study from which a NOAEL can be derived was needed for the safety evaluation of these substances.

In this revision of FGE.74, FGE.74Rev4 the Panel evaluated a new dossier addressing this issue. The dossier contains repeated-dose subchronic toxicity studies on the representative substance, methyl propyl trisulfide [FL-no: 12.020] to cover the evaluation of the substances in the tri- and polysulphides subgroup (subgroup VI).

The Panel noted that [FL-no: 12.020] is reported as a mixture of 45% purity, with 54% mono-, di-and tri-sulphides as secondary components (see Table2). The material tested in the repeated-dose toxicity studies has a higher purity (~ 70%). Thirty per cent of the tested preparation has not been identi_{fied. Regarding the higher purity of the substance tested, the Panel considers that the potential} risk associated with the secondary components present in commercial preparations might have been underestimated in the performed toxicity study.

In the 14-day dose-range finding repeated-dose toxicity study (see Section6.1), the recovery of the administered substance [FL-no: 12.020] in the feed decreased from 75% to 50% within a week (Bauter, 2015a). Therefore, the applicant decided to perform the 90-day study by gavage (see Section 6.3). It is unknown whether this disappearance of the substance from feed is due to volatilisation or reaction to potentially more toxic products in food. Without knowing the fate of the missing fraction, it cannot be assessed whether these products would be covered by a gavage study.

6.

Short-term and subchronic toxicity

6.1.

A 14-day range-

finding study with methyl propyl trisulfide [FL-no:

12.020] mixed in the diet (Bauter, 2015a)

In an initial experiment, the candidate substance was administered via the feed to male and female rats during 14 days (Bauter, 2015a) at levels targeting 150, 300 and 600 mg/kg bw per day. However, the compound was not stable in the diets so that the recovery of the administered substance [FL-no: 12.020] in the feed decreased from 75% to 50% within a week. Although none of the rats died, after autopsy enlarged spleens with dark discoloration at all dose levels was observed. This indicates haemolysis, a known effect of sulphides. Because of the low stability of the compound in the feed, as well as the poor palatability, the company decided to administer it by oral gavage in a follow up experiment, at lower doses.

For a summary of the additional toxicity studies considered by EFSA, see AppendixD, TableD.1.

6.2.

A 14-day range

finding study with methyl propyl trisulfide [FL-no:

12.020] by oral gavage (Bauter, 2015b)

A 14 day range-finding study by oral gavage was performed in male and female Sprague–Dawley rats (5/sex per dose) at dose levels 12.5 (Group 2), 50 (group 3) and 100 (group 4) mg/kg bw per day (Bauter 2015b). The test substance was mixed with corn oil.

There were no test substance-related mortalities. No clinical observations or changes in body weight, body weight gain, mean daily food consumption or food efficiency associated with test substance administration were observed.

Blood cytology evaluation revealed the presence of Heinz bodies in all Groups 3 and 4 animals; these were present with a lower incidence and severity in Group 2 animals. Additionally, Group 4 animals had evidence of polychromasia. Splenic enlargement and/or dark red discoloration of the spleen were observed in all Groups 3 and 4 animals and in individual Group 2 males and females. Spleen weights were increased. Microscopic findings of increased spleen iron deposits and evidence of increased splenic erythropoiesis were observed in Groups 3 and 4 and/or individual Group 2 males and females. For a study of longer duration, by means of oral gavage administration of the test substance animals were expected to tolerate dose levels up to 12.5 mg/kg bw per day.

For a summary of the additional toxicity studies considered by EFSA, see AppendixD, TableD.1.

6.3.

A 90-day study by oral gavage with [FL-no: 12.020]

During a 90-day subchronic oral gavage study according to OECD guideline 408 and Good laboratory practice (GLP), Sprague–Dawley CDâ rats (10/sex per Group) received 0.5, 2 and 6 mg/kg bw per day of the test substance mixed with corn oil (0.1, 0.4, and 1.2 mg/mL, w/v, respectively) by

oral gavage; vehicle controls received corn oil (5 mL/kg bw per day) (Koetzner, 2016). The test substance was shown to be stable in the solutions for gavage for the whole period.

There were no mortalities or changes in clinical/ophthalmological parameters, body weight, body weight gain, food consumption, or food efficiency attributable to methyl propyl trisulfide administration. There were no test substance-related changes in most male and female haematology, coagulation, clinical chemistry and urinalysis values. Small decreases in red blood cell count, haemoglobin and haematocrit observed in the high-dose groups, in males and females. These effects are consistent indications of haematotoxic effects and are considered adverse.

There were no macroscopic or microscopic changes attributable to administration of the test substance. No differences in organ weights, organ-to-body weight ratios or organ-to-brain weight ratios were observed in female rats. Decreases in absolute thymus weights in high-dose male rats were without histologic correlates and were not directly correlated with any other study parameters and are, therefore, not considered adverse.

Under the conditions of the study, the NOAEL of the test compound was determined to be 2 mg/kg bw per day for both males and females based on haematotoxic effects.

For a summary of the additional toxicity studies considered by EFSA, see AppendixD, TableD.1.

6.4.

EFSA considerations

The Panel noted the uncertainties regarding the suitability of the flavouring substance [FL-no: 12.020] administered in the toxicity study to represent the material of commerce, and the potential formation of reaction products in feed, as outlined in Sections 5 and 6.1. Despite EFSA’s request, the applicant did not provide the respective information.

Therefore, the Panel decided that the 90-day study can be considered only once it is clearly demonstrated that the material tested is representative of the material of commerce and that potential reaction products are not of safety concern.

7.

Application of the Procedure

7.1.

Application of the Procedure to 19 aliphatic sulphides and thiols

evaluated by the JECFA

9

(JECFA, 2000, 2004b)

According to JECFA, 15 substances belong to structural class I and 4 to structural class II using the decision tree approach presented (Cramer et al., 1978).

None of the substances could be anticipated to be metabolised to innocuous products and all were evaluated via the B-side of the Procedure. The estimated daily per capita intakes of the 19 flavouring substances are below the threshold of concern for structural class I and II, and a NOAEL exists to provide an adequate MoS to the estimated intake asflavouring substances (Step B4).

Step B4

For erythro- and threo-3-mercapto-2-methylbutanol [FL-no: 12.291] (3-mercapto-2-methyl-1-butanol)], the no observed effect level (NOEL) of 0.7 mg/kg bw per day for the structurally related substance 2-mercapto-3-butanol [FL-no: 12.024] from a 92-day study in rats fed by gavage (Cox et al., 1974) provides an adequate MoS (> 10,000) in relation to known levels of intake of this agent.

This NOEL is also appropriate for the structurally related agents ( )-2-mercapto-2-methylpentan-1-ol [FL-no: 12.241], methylpentan-1-)-2-mercapto-2-methylpentan-1-ol (racemic) [FL-no: 12.238], 3-mercapto-2-methylpentanal [FL-no: 12.239] and ()-ethyl 3-mercaptobutyrate [FL-no: 12.255], because they are all acyclic thiols with oxidised side-chains that are anticipated to undergo oxidation or hydrolysis and subsequent metabolism via similar metabolic pathways.

For 4-mercapto-4-methyl-2-pentanone [FL-no: 12.169], the NOEL of 1.9 mg/kg bw per day for the structurally related substance 3-mercapto-2-pentanone [FL-no: 12.031] administered to rats by gavage in a 92-day study (Morgareidge, 1971) provides an adequate MoS (> 10,000) in relation to known levels of intake of this agent.

For ethyl 4-(acetylthio)butyrate [FL-no: 12.257], the NOEL of 6.5 mg/kg bw per day reported in a 13-week study in rats (Shellenberger, 1970) fed with the structurally related substance ethylthioacetate [FL-no: 12.018] provides an adequate MoS (> 10,000) in relation to known levels of intake of this agent.

9 _{The text from the indicated reference sources was taken verbatim with the only difference that the JECFA numbers were}

For 2-(methylthio)ethanol [FL-no: 12.179], the NOEL of 1.4 mg/kg bw per day reported in a 13-week study in rats (Cox et al., 1979) fed by gavage with the structurally related substance 2-(methylthiomethyl)-3-phenylpropenal [FL-no: 12.087] provides an adequate MoS (> 10,000) in relation to known levels of intake of this agent. This NOEL is also appropriate for the structurally related agent ethyl-5-(methylthio)valerate [FL-no: 12.212], which is also an acyclic sulfide with an oxidised side-chain that is anticipated to undergo oxidation and subsequent metabolism via similar pathways.

For 2,3,5-trithiahexane [FL-no: 12.198], the NOEL of 0.3 mg/kg bw per day reported in a 13-week study (Mondino, 1981) in rats fed with the structurally related substance 3-methyl-1,2,4-trithiane [FL-no: 15.036] provides an adequate MoS (> 10,000) in relation to known levels of intake of this agent.

For diisopropyl trisulphide [FL-no: 12.280], the NOEL of 4.8 mg/kg bw per day reported in a 13-week study (Morgareidge and Oser, 1970a) in rats fed by gavage with the structurally related substance dipropyl trisul_{fide [FL-no: 12.023] provides an adequate MoS (> 100,000) in relation to} known levels of intake of this agent.

For diallyl trisulfide [FL-no: 12.009] and dipropyl trisulfide [FL-no: 12.023], the NOELs of 4.6 and 4.8 mg/kg bw per day, respectively, were reported in a 90 days study (Morgareidge and Oser, 1970a, b) at a single dose, which gave adequate margins of safety for [FL-no: 12.013, 12.020, 12.045, 12.074 and 12.155]. The dose that had no effect is more than 10,000 times greater than the estimated per capita intake in Europe and more than 100,000 times higher than the estimated per capita intake in the United States.

No adequate NOEL was available for diallyl sulfide [FL-no: 12.088] or a related substance, therefore no adequate MoS can be provided. Accordingly, the evaluation of the substance proceeds to Step B5. Step B5

For diallyl sulfide [FL-no: 12.088], the intake is estimated to be 0.4 lg/capita per day in the USA, which is lower than 1.5 _{lg/day, therefore, JECFA has concluded that there is no safety concern based} on the intake data.

In conclusion, JECFA evaluated all substances as to be of no safety concern at the estimated levels of intake asflavouring substances based on the MSDI approach.

The evaluations of the 19 aliphatic sulfides and thiols with the outcome of the JECFA evaluations are summarised in Appendix E, TableE.1of this FGE.

7.2.

Application of the Procedure to aliphatic and alicyclic mono-, di-,

tri-, and polysulphides with or without additional oxygenated

functional groups evaluated by EFSA in FGE.08Rev5 (EFSA CEF

Panel, 2012b)

10

The application of the Procedure is based on intakes estimated on the basis of the MSDI approach. For the candidate substance methyl methanethiosulfonate [FL-no: 12.159] (the only substance in subgroup X), there is an indication of a genotoxic potential in vitro. Furthermore, for three candidate substances (in subgroup III), 2-methylbutane-2-thiol [FL-no: 12.172], 2-methylpropane-2-thiol [FL-no: 12.174] and ethyl-2-mercapto-2-methyl propanoate [FL-no: 12.304] and one candidate substance (in subgroup VII), 2,4,4-trimethyl-1,3-oxathiane [FL-no: 16.057], a concern for genotoxicity was also identified based on experimental evidence for [FL-no: 12.174] and the structural similarity among these four substances. Therefore, in the absence of further genotoxicity data, the Panel concluded that the Procedure could not be applied to these five substances.

For four candidate substances, 3-mercaptooctanal [FL-no: 12.268] (subgroup III), 3-mercaptodecanal [FL-no: 12.269] (subgroup III), methanedithiol diacetate [FL-no: 12.271] (subgroup VIII) and 3,5-dimethyl-1,2-dithiolane-4-one [FL-no: 12.295] (subgroup V), no data on use as flavouring substances in Europe are available. Therefore, no intakes in Europe can be estimated and accordingly the Panel concluded that the Procedure could not be applied to these four substances.

Thus, for in total nine candidate substances, the Procedure could not be applied: [FL-no: 12.159, 12.172, 12.174, 12.268, 12.269, 12.271, 12.295, 12.304 and 16.057].

For the safety evaluation of the remaining 71 candidate substances from chemical groups 20 and 30 the Procedure as outlined in Annex I was applied, based on the MSDI approach. The stepwise evaluations of the 71 substances evaluated through the Procedure are summarised in TableE.1.

10 _{The text is taken verbatim from the indicated reference source, but text related to subgroups not included in the present FGE}

Step 1

The candidate substances were classified following the procedure established by Cramer et al. (1978). For the 71 candidate substances evaluated through the Procedure, 42 substances were classi_{fied into structural class I, 19 substances were classified into structural class II and 10 substances} were classified into structural class III.

Step 2

Step 2 requires consideration of whether metabolic pathways exist to metabolise the candidate substances to innocuous products at the expected levels of intake. The candidate substances may be biotransformed to reactive metabolites, such as thiols, sulphoxides and sulphones and, in consequence, they are not predicted to be metabolised to innocuous products. Therefore, the evaluation of all 71 candidate substances proceeds via the B-side of the Procedure scheme.

Step B3

The 42 substances in structural class I have estimated European daily per capita intakes ranging from 0.0012 to 6.1 lg, which is below the threshold of concern of 1,800 lg/person per day. The 19 substances evaluated through the Procedure in structural class II have estimated European daily per capita intakes ranging from 0.0024 to 2.4 _{lg, which is below the threshold of concern for class II of} 540 _{lg/person per day. The 10 substances in structural class III have estimated European daily per} capita intakes from 0.012 to 6.1_{lg, which is below the threshold of concern for class III of 90 lg/person} per day. Accordingly, all 71 candidate substances proceed to Step B4 of the Procedure.

Step B4

No adequate studies on candidate substances are available. Repeated-dose toxicity studies are available on some supporting substances, which, with very few exceptions, have been carried out testing only one dose, giving rise to no observed adverse effects. The results of the adequate studies on supporting substances show a relatively high degree of variability in the reported NOAELs, ranging from 0.06 to 250 mg/kg bw per day.

The 20 candidate substances in subgroup I can be represented by the supporting substance dimethyl sulfide [FL-no: 12.006], for which an adequate 90-day subchronic study is available, indicating that no adverse effects were produced by the highest oral dose tested (250 mg/kg bw per day), which can be considered a NOAEL. The combined estimated daily per capita intake of 10 lg for the 18 candidate substances in subgroup I corresponds to 0.17 _{lg/kg bw per day at a body weight of} 60 kg. Thus, a MoS of 1.5 _{9 10}6 _{can be calculated. The 20 candidate substances in subgroup I are} accordingly not expected to be of safety concern at the estimated levels of intake.

Within subgroup III, adequate 90-day subchronic studies are available for four supporting secondary thiols, 2-mercapto-3-butanol [FL-no: 12.024], cyclopentanethiol [FL-no: 12.029], 2,3- and 10-mercaptopinane [FL-no: 12.035] and 2,6-(dimethyl)thiophenol [FL-no: 12.082], which can be considered representative of the 11 candidate substances evaluated through the Procedure in this subgroup. In the four studies, no adverse effects were produced by the highest oral dose tested ranging from 0.06 up to 0.7 mg/kg bw per day. By adopting a conservative approach, the lowest value (0.06 mg/kg bw per day) can be considered a NOAEL. The combined estimated daily per capita intake of 1.13 lg for the 11 candidate substances evaluated through the Procedure in subgroup III corresponds to 0.019 lg/kg bw per day at a body weight of 60 kg. Thus, a MoS of 3 9 103 can be calculated. The 11 candidate substances in subgroup III are accordingly not expected to be of safety concern at the estimated levels of intake.

Within subgroup V, adequate 90-day subchronic studies are available for two supporting substances dicyclohexyl disul_{fide [FL-no: 12.028] and benzyl methyl disulfide [FL-no: 12.068], which can be} considered representative of the four candidate substances in this subgroup evaluated through the Procedure. In the two studies, no adverse effects were produced by the highest oral dose tested: 0.23 and 1.15 mg/kg bw per day. By adopting a conservative approach, the lowest value (0.23 mg/kg bw per day) can be considered a NOAEL. The combined estimated daily per capita intake of 0.6 lg for the four candidate substances evaluated through the Procedure in subgroup V corresponds to 0.01 lg/kg bw per day at a body weight of 60 kg. Thus, a MoS of 2.3 9 104 can be calculated. The four candidate substances in subgroup V are accordingly not expected to be of safety concern at the estimated levels of intake.

Within subgroup VI, no adequate toxicity study from which a NOAEL could be established was available, neither on the candidate substances nor on supporting substances. Therefore, the

Panel concluded that additional data are still required for the eight tri-, tetra- and polysulphides in subgroup VI of FGE.08 [FL-no: 12.093, 12.094, 12.097, 12.100, 12.112, 12.116, 12.164 and 12.167].11 Within subgroup VIII, an adequate 90-day subchronic study is available for one supporting substance, ethyl thioacetate [FL-no: 12.018], which can be considered representative of the eight candidate substances evaluated through the Procedure in this subgroup. In the study, no adverse effects were produced by the highest oral dose tested: 6.63 mg/kg bw per day. Therefore, the NOAEL is concluded to be 6.63 mg/kg bw per day for ethyl thioacetate. The combined estimated daily per capita intake of 2.4 lg for the eight candidate substances in subgroup VIII corresponds to 0.04 lg/kg bw per day at a body weight of 60 kg. Thus, a MoS of 1.7 9 105 can be calculated. The eight candidate substances in subgroup VIII are accordingly not expected to be of safety concern at the estimated levels of intake.

The conclusion from Step B4 is that for the 43 candidate substances belonging to subgroups I, III, V and VIII, and evaluated through the Procedure, adequate NOAELs exist for the candidate substance or for structurally related substances providing adequate margins of safety at the estimated levels of intake. Therefore, these candidate substances are not expected to be of safety concern at the levels of exposure estimated by the MSDI approach.

For the eight candidate substances belonging to subgroup VI [FL-no: FL-no: 12.093, 12.094, 12.097, 12.100, 12.112, 12.116, 12.164 and 12.167], additional toxicity data are required.

The evaluations of the aliphatic and alicyclic mono-, di-, tri- and polysulphides are summarised in Appendix E, TableE.2.

7.3.

EFSA Considerations relevant for FGE.74

The 19 JECFA-evaluated aliphatic sulphides and thiols are distributed into five subgroups of structurally related substances. The subgrouping is in compliance with the one used in FGE.08Rev5 (see Section 2.2.1and Table 1).

Although the available data are limited, the Panel considered that for the remaining 19 substances in FGE.74Rev4 the genotoxicity data do not preclude evaluating these substances through the Procedure.

The Panel agrees with the application of the Procedure as performed by JECFA for five aliphatic sulphides and thiols, namely [FL-no: 12.238, 12.239, 12.255, 12.257 and 12.291].

For 14 substances [FL-no: 12.009, 12.013, 12.020, 12.023, 12.045, 12.074, 12.088, 12.155, 12.169, 12.179, 12.198, 12.212, 12.241 and 12.280], the Panel did not agree with the application of the Procedure by JECFA for the following reasons:

JECFA derives a NOAEL of 1.4 mg/kg bw per day reported in a 13-week study in rats (Cox et al., 1979) fed by gavage with 2-(methylthiomethyl)-3-phenylpropenal [FL-no: 12.087]. The Panel did not agree with JECFA that 2-(methylthiomethyl)-3-phenylpropenal [FL-no: 12.087] is structurally related to 2-(methylthio)ethan-1-ol [FL-no: 12.179] or ethyl-5-(methylthio)-valerate [FL-no: 12.212]. JECFA derived a NOAEL of 0.3 mg/kg bw per day reported in a 13-week study (Mondino, 1981) in rats fed with 3-methyl-1,2,4-trithiane [FL-no: 15.036]. The Panel does not agree with JECFA that 2,3,5-trithiahexane [FL-no: 12.198] is structurally related to 3-methyl-1,2,4-trithiane [FL-no: 15.036].

However, in the first revision of FGE.74, FGE.74Rev1, all substances have been distributed to subgroups with respect to sulphur-containing functional groups, according to FGE.08 and following revisions. The JECFA-evaluated substances 2-(methylthio)ethan-1-ol and ethyl-5-(methylthio)valerate [FL-no: 12.179 and 12.212] have been allocated to subgroup I, Acyclic sulphides, and 2,3,5-trithiahexane [FL-no: 12.198] has been allocated to subgroup V, Acyclic and cyclic disulphides. Appropriate NOAELs exist for these two subgroups, as is argued in FGE.08Rev5. Since based on these NOAELs adequate margins of safety can be calculated for [FL no: 12.179, 12.198 and 12.212], in line with JECFA, the Panel also concludes that these substances are not expected to be of safety concern at the estimated levels of intake.

For the diallyl sulfide [FL-no: 12.088], JECFA evaluated this substance at Step B5 of the Procedure to be of no safety concern as the estimated intake in the USA of 0.4 lg/capita per day is below 1.5lg/person per day. In line with the opinion expressed by the Scientific Committee on Food (SCF, 1999), the Panel does not make use of this threshold of 1.5 lg/person per day. However, this substance is allocated into subgroup I, for which an appropriate NOAEL (reported for dimethylsulfide [FL-no: 12.006]) exist, as is demonstrated in FGE.08Rev5. The NOAEL of 250 mg/kg bw per day

11